A wide variety of commercially important commodity or specialty chemicals are produced through metabolic engineering, the manipulation of living organisms to achieve desirable metabolic substrates, products and/or byproducts. The advent of recombinant DNA technology has enabled metabolic pathway modification using targeted genetic modifications instead of and/or in addition to the traditional mutagenesis and selection approach. Since the mid 1980s, numerous examples of metabolic engineering have been reported. In addition to manipulating the genetic code, variables such as temperature and aerobic conditions can also be engineered or manipulated to achieve the desired results. One of the major limitations in such bio-production processes is the toxic effect of many of the desired substrates, products and/or byproducts. This is a problem of both specific and general significance because it is encountered in the production of commodity chemicals from renewable sources, bioremediation technologies, and the use of cells in biocatalysis involving toxic organic molecules.
The strictly anaerobic, Gram-positive, solventogenic clostridia (C. acetobutylicum, C. beijerinckii and related species) are excellent candidates for generating metabolically engineered strains for several potential applications. The fermentation byproducts of these species may lead to industrial processes for production of butanol, butyric acid, acetone, butanediol, propanol, 1,3-propanediol, polysaccharides, and enzymes, or for biotransformations and bioremediation. For example, clostridia grow under a low redox potential, enabling a variety of stereospecific reductions yielding chiral products that are difficult to synthesize chemically. In addition, these and related clostridial species can degrade a number of toxic chemicals and are thus good candidates for bioremediation applications. A major advantage of solventogenic clostridia is their ability to utilize an unusually large variety of substrates: mono-, oligo- and poly-saccharides, including the most common pentoses and hexoses, and as such to utilize biomass hydrolysates.
Important potential applications of solventogenic clostridia include production of butanol and acetone. However, such production is affected by poor process economics; for example, a typically low butanol titer in the product stream. Low butanol titers are due to the low tolerance of these organisms to butanol, with final butanol concentrations rarely exceeding 12-13 g/l. As a result, butanol separation costs are high. Economic analyses show that if the final butanol concentration was raised from 12 to 19 g/l, the separation costs would be cut in half.
As mentioned above, butanol toxicity is quite severe. Butanol concentration in the final product stream usually cannot exceed 12-13 g/l, before cellular degradation. At high concentrations, butanol inhibits active nutrient transport, membrane bound ATPase, glucose uptake, partially or completely abolishes the membrane ΔpH and Δψ, and lowers the intracellular pH. To date, butanol toxicity has been attributed to its chaotropic effect on the cell membrane. However, the membrane model may not afford a complete explanation for butanol toxicity, as a clostridia strain with the inactive buk gene produces 230 mM (17 g/l) butanol, 83 mM (4.8 g/l) acetone and 69 mM (3.2 g/l) ethanol (total solvents of 25 g/l) [Harris, L. M., Desai, R. P., Welker, N. E., Papoutsakis, E. T. “Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?”, Biotechnol. Bioeng., 67: 1-11 (2000)]. This strain overcame the 12-13 g/l butanol-toxicity limit without any specific selection or adaptation for increased butanol tolerance.
Stress-response proteins are a collection of specialized proteins that are essential to cellular function and are present in non-stressed cells under normal growth conditions, playing an important role in cell physiology as a protective cellular response to environmental stress. A common family of these stress response proteins are termed heat shock proteins (HSPs) due to their abundance following heat shock. The stress response proteins bind normative states of other proteins and assist in proper folding by recognizing exposed hydrophobic surfaces on normative protein species, which ultimately end up buried when the protein is in its properly folded, functional state. Generally, stress response proteins form noncovalent interactions with the hydrophobic regions of misfolded proteins, thereby stabilizing them from irreversible multimeric aggregation, misfolding of nascent polypeptides, unfolding during exposure to stress and eventual degradation. The stabilized and properly folded proteins are therefore available to perform their cellular function(s).
The major heat shock protein classes are the 90-kDa heat shock protein (HSP90), the 60-kDa heat shock protein (HSP60; including GroEL), the 70-kDa heat shock protein (HSP70; DnaK in E. coli) and 40-kDa heat shock protein (HSP40 or the DnaJ family). Another important protein involved in the heat shock response is a co-chaperone of HSP60 called chaperonin 10 (cpn10; GroES in E. coli).
DnaK operates by binding to nascent polypeptide chains on ribosomes, preventing premature folding, misfolding, or aggregation. DnaK is composed of two major functional domains. The NH2-terminal ATPase domain and the COOH-terminal domain. The NH2-terminal ATPase domain binds ADP and ATP and hydrolyzes ATP, whereas the COOH-terminal domain is responsible for polypeptide binding. DnaJ is a co-chaperone for DnaK. GrpE is another chaperone involved in the DnaKJ folding pathway by facilitating the exchange between ADP and ATP. The genes for DnaK, DnaJ and GrpE are organized as an operon (the dnaK operon).
Another class of HSPs is the GroEL/ES family of proteins that bind partially folded intermediates, preventing their aggregation, and facilitating folding and assembly. In addition, it has been suggested that GroEL, with the assistance of its co-chaperonin GroES, may allow misfolded structures to unfold and refold. The GroEL of E. coli consists of 14 identical subunits in two-stacked heptameric rings, each containing a central cavity. The size of the GroEL/ES complex cavity suggests that proteins of up to 50-60 kDa can be handled by this chaperone system. The genes for GroEL/ES are also organized as an operon (the groE operon). In B. subtilis, expression of the dnaK and groE operons is negatively regulated by a repressor protein through a CIRCE DNA element (a palindromic sequence between the promoter and the initiation codon). For example, in B. subtilis inactivation of this repressor protein (HrcA)—whose activity is modulated by GroEL/ES—results in constitutive expression of the two HSP operons, and this enhances the folding and secretory production of proteins which are difficult to fold.
HSPs have also been detected in solventogenic clostridia. Terracciano, et al. showed that a moderate increase in butanol concentration can cause a response in C. acetobutylicum similar to that obtained from heat shock. Using a chemostat, Pich, et al. demonstrated that the synthesis rates of HSPs, as well as solventogenic proteins, increased several hours before solvents were detected in the medium. It was also noted by Bahl that some of the factors necessary for the initiation of solventogenesis in C. acetobutylicum may also be responsible for the induction of stress response, namely lowered pH, low growth rate, and excess carbohydrates.
The preceding studies only allude to the presence of heat/shock stress response proteins in solventogenic bacteria, but without conclusive evidence regarding the role of such proteins or guidance as to their use in solvent production. The viability of solventogenic bacteria has been a long-standing, unresolved concern of the prior art. There remains a need in the art to provide an organism and/or related method to enhance fermentation by such bacteria and increase solvent production.
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